title: A novel approach to quantitative LC-MS/MS: therapeutic drug monitoring of clozapine and norclozapine using isotopic internal calibration.
abstract: Therapeutic drug monitoring (TDM) requires timely results in order to be clinically helpful. Such assays, when carried out using mass spectrometry-based methods, typically involve a batched sample approach with multipoint calibration. Isotopic internal calibration offers the possibility of open-access mass spectrometric analysis with consequent shortening of turnaround times. We measured plasma clozapine and N-desmethylclozapine (norclozapine) concentrations in (1) external quality assessment (EQA) samples (N = 22) and (2) patient samples (N = 100) using liquid chromatography-tandem mass spectrometry with isotopic internal calibration (ICAL-LC-MS/MS). Analyte concentrations were calculated from graphs of the response of three internal calibrators (clozapine-D4, norclozapine-D8, and clozapine-D8) against concentration. Precision (% RSD) and accuracy (% nominal concentrations) for the ICAL-LC-MS/MS method were <5 % and 104-112 %, respectively for both analytes. There was excellent agreement with consensus mean and with 'spiked' values on analysis of the EQA samples (R (2) = 0.98 and 0.97, respectively, inclusive of clozapine and norclozapine results). In the patient samples, comparison against traditionally calibrated HPLC-UV and LC-MS/MS methods showed excellent agreement (R (2) = 0.97 or better) with small albeit significant mean differences (<0.041 and <0.042 mg/L for clozapine and norclozapine, respectively). These differences probably reflect discrepancies in the in-house preparation of calibrators and/or interference in the UV method. Internal calibration offers a novel and attractive alternative to traditionally calibrated batch analysis in analytical toxicology. The method described has been validated for use in the high-throughput TDM of clozapine and norclozapine, and allows for (1) same-day reporting of results and (2) significant cost savings.
authors.affiliation: Toxicology Unit, Department of Clinical Biochemistry, King's College Hospital NHS Foundation Trust, London, SE5 9RS, UK, lewis.couchman@nhs.net.
authors.forename: Lewis
authors.initials: L
authors.lastname: Couchman
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Belsey
authors.forename: Simon A
authors.initials: SA
authors.lastname: Handley
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Flanagan
mesh_terms: D014150:Antipsychotic Agents / Q000097:blood* / Q000627:therapeutic use; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D003024:Clozapine / Q000031:analogs & derivatives* / Q000097:blood* / Q000627:therapeutic use; D003903:Deuterium / Q000737:chemistry; D016903:Drug Monitoring / Q000379:methods*; D004358:Drug Therapy; D006801:Humans; D007553:Isotope Labeling; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
chemical_list: D014150:Antipsychotic Agents; C058272:norclozapine; D003903:Deuterium; D003024:Clozapine